Recovery of nitrogenous material from micro-organisms

ABSTRACT

Cells of a micro-organism are treated with a proteolytic enzyme to cause degeneration of protein components of the cell wall, thereafter deactivating or removing the proteolytic enzyme and continuing enzymatic attack on the cell wall with a carbohydratase (whereby attack on the protein of the cytoplasm is avoided or reduced) thereafter recovering an aqueous fraction containing nitrogeneous material.

United States Patent t Corteel 1 Feb. 29, 1972 [54] RECOVERY OFNITROGENOUS I [56] H MATERIAL FROM MICRO-ORGANISMS, UNITEDSTATES PATENTS[721 Philip!e 3,088,879, 5/1969 Weaver .195/2 [73] Assignee: The BritishPetroleum Company Limited, 3, 7 4/1965 Weaver... .195/2 London, EnglandI 3,330,735 7/1967 Napier. ..l95/5 [22] Filed: Apr. 13, 1967 PrimaryExaminer-Lionel M. Shapiro [21] PP N04 630,510 Attorney-Morgan,Finnegan, DurhamandPin'e 30 Foreign Application-Priority mm [511 vABSTRACT Apr. 15,1966 Great Britain.....- ..-....l6,536/66 Cells of amicro-organism are treated with a proteolytic enzyme to causedegeneration of protein components of the cell U-S- "195/4, wallthereafter deactivating or removing the proteolytic en- [51] cud 13/zyme and continuing enzymatic attack on the cell wall with a [58] Fieldof Search ..l95/2, 4, 5,29, carbohydratase (whereby attack on theprotein of the cytoplasm is avoided or reduced) thereafter recovering anaqueous fraction containing nitrogeneous material.

9 Claims, No Drawings RECOVERY OF NITROGENOUS MATERIAL FROMMHCRQ-ORGANHSMS This invention relates to a process for the recovery ofnitrogeneous material from micro-organisms.

Processes are well known for the rupture of the cell walls ofmicro-organisms. While these methods are efficient for the rupture ofthe cell walls they have defects which detract from their value as abasis for a commercial process for the recovery of cytoplasmicnitrogeneous material from micro organisms.

A process has been proposed for the rupture of cell walls by means of anenzyme which attacks carbohydrate components of compounds, present inthe cell wall, in which these components form a part. Thus, it haspreviously been proposed, a beta-glucanase may be employed to attackcellulose and mannan structures in the cell wall.

in distinction to this previously proposed process there is now provideda process in which, in an initial enzymic step, a micro-organism istreated with a proteolytic enzyme, whereby compounds of carbohydrate andprotein in the cell wall are attacked and thereafter the weakened cellwall is attacked by means of an enzyme which attacks a carbohydratecomponent of the cell wall, whereby the cell wall is further weakened oris ruptured.

Thus according to the present invention there is provided a processwhich comprises treating with a proteolytic enzyme a material whichconsists of or contains cells of a micro-organism, said cells having acell wall, constructed at least in part of at least one compound havinga carbohydrate component and a protein component, the treatment of saidmaterial being carried out whereby degeneration of the protein componenttakes place and whereby the carbohydrate component is renderedsusceptible to enzymic attack, thereafter deactivating or removing theproteolytic enzyme which is present in the cell walls, thereafterfurther treating said material, or a fraction containing the firsttreated micro-organism which has been recovered therefrom, with anenzyme, capable of attacking a carbohydrate present in the cell wall,whereby degeneration of said carbohydrate takes place and thereafter,with or without an intermediate step for the further attack on orrupturing of the cell wall, recovering an aqueous fraction containingnitrogeneous compounds in solution and/r suspension.

Within the term micro-organism used herein we include mixtures ofmicro-organisms.

Micro-organisms which are treated as herein described may be yeasts,moulds or bacteria.

The bacteria referred to in this specification are classified accordingto the system described in Bergeys Manual of Determinative Bacteriologyby R. S. Breed, E. G. D. Murray and N. R. Smith (1957) 7th Edition,published by Williams (Baltimore, U.S.A.). The yeasts are classifiedaccording to The Yeasts-a Taxonomic Study by Lodder and Kreger-van Rij(1952) published by North Holland Publishing Company (Amsterdam).

Preferably when a yeast is employed this is of the familyCryptococcaceae and particularly of the subfamily Cryptococcoideae;however, if desired there may be used, for example, ascosporogeneousyeasts of the subfamily Saccharomycoideae. Preferred genera of theCryptococcoideae subfamily are Torulopsis (also known as Torula),Candida and Mycoderma. Preferred species of yeast are as follows. Inparticular it is preferred to use the specific stock of indicatedreference number; these reference numbers refer to CBS stock held by theCentraal Bureau vor Schimmelculture, Baarn, Holland and to lMRA stockheld by the Institut National de la Recherche Agronomique, Paris,France.

Preferred strain Candida lipolytica CBS 599 Candida pulcherrima CBS 6l0Candida utilis CBS 890 Candida utilis, Variati major CBS 841 Candidatropicalis CBS 2317 Candida arborea Torulopsis colliculosa CBS 133Hansenula anomala CBS Oidium lactia Neurospora sitophila Mycodermacancoillote INRA; STV ll Of the above Candida lipolytica is particularlypreferred.

Suitably there are used micro-organisms capable of growth on a straightchain paraffinic hydrocarbon. Preferably there are used micro-organismswhich have been grown on a hydrocarbon containing substrate. Suitablesubstrate include kerosene, gas oils and lubricating oils; thesefeedstocks may be unrefined or may have undergone some refinerytreatment, but will usually be required to contain a proportion ofstraight chain hydrocarbons in order to fulfill the purpose of thisinvention. Suitably there isused a petroleum fraction containing 3-45percent by weight of straight chain hydrocarbons.

Preferably a petroleum fraction is used consisting at least in part ofstraight chain hydrocarbons and having a mean molecular weightcorresponding to at least 10 carbon atoms per molecule.

Preferred methods for use in the cultivation of the micro-organism andfor the recovery of the product are described in British Patentspecification Nos. 914,567, 914,568, 1,017,584 and 1,017,585-also inBritish Patent application No. 44,606/62.

Preferably the proteolytic enzyme employed in the process of theinvention is an enzyme having activity for breaking only a bond betweentwo specific amino acids present in a protein component of the cellwall, whereby the extent of degradation of the protein component isseverely limited. Furthermore, since some penetration of the cytoplasmby the proteolytic enzyme may occur, the sue of an enzyme with thislimited activity is also desirable since it follows that any degradationof protein of the cytoplasm will be limited in similar manner.

Suitable proteolytic enzymes for use in the process include pepsin,trypsin, bromelin, ficin and papain.

The use of papain is preferred. Suitably the amount of papain employedis 0.2-0.3 percent by weight of dry cell weight.

Preferably, the proteolytic enzyme is employed under conditions suchthat a substantial proportion of the cellular nitrogeneous component issolubilized.

Preferably, however, the conditions under which the proteolytic enzymeis employed are also such that only a minor proportion of thecytoplasmic proteins is degraded, i.e., converted into low molecularweight polypeptides or into amino acids.

It is an important feature of the invention that the proteolytic enzymeis deactivated or removed after use and before treatment with an enzymewhich attacks the carbohydrate of the cell. As a result of the attack onthe carbohydrate, the protein of the cytoplasm is rendered morevulnerable to attack by a proteolytic enzyme and the rate of attack onprotein of said type would be severe; it is for this reason that thedeactivation or removal of the proteolytic enzyme at the appropriatestage is important. Furthermore, even in the first stage enzymaticattack on the cell wall, some attack on cytoplasmic protein will takeplace and for this reason also the period of contact of the cell withthe proteolytic enzyme before removal or destruction of said enzymeshould be limited to the period necessary for effecting the attack onthe cell wall protein.

Preferably the determination of suitable conditions for the use of theproteolytic enzyme is carried out as follows: i

After determination of the total cellular nitrogen, the sample ofmicro-organisms is treated in a series of tests, with increasing amountsof enzyme.

The total solubilized nitrogen is then estimated and the following ratiois calculated:

The concentration of enzyme employed will be the minimum concentrationcorresponding to the maximum of this ratio.

Preferably the treated material remaining after the treatment with theproteolytic enzyme is subjected to phase separation and if desired theaqueous phase so recovered, with or without intervening treatment, maybe reemployed or recycled for the treatment of further amounts of themicro-organism. Phase separation is preferably by centrifuging. Afterphase separation the micro-organism-containing fraction is preferablywashed with an aqueous medium, suitably water, to remove the proteolyticenzyme, at least insofar as the enzyme is associated with the cellwalls.

The washed micro-organism may be subjected to a further phaseseparation, suitably by centrifuging.

The micro-organism, having been treated to remove or destroy proteolyticenzyme associated with the cell wall, should be treated with an enzymecapable of attacking carbohydrates in the next treating stage withoutundue delay and certainly before there is time for the destruction ofthecells by autoiysis.

In this further treating stage at least some of the carbohydratecomponents of the cell wall, for example cellulose and mannan, areattacked. Since the enzymes which are employed are not proteolytic it ispossible to operate under severe conditions without substantialdegradation of protein of the cytoplasm. If desired a lipase may beemployed, simultaneously or subsequently, to effect a further or morerapid weakening of the cell wall.

After treatment with enzymes for the degradation of the carbohydrates ofthe cell wall and also if desired for the degradation of lipids, theaqueous phase will contain a substantial proportion of the protein ofthe cytoplasm in solution or suspension.

The aqueous phase with colloidally suspended protein may be separated,for example by centrifuging.

If desired this aqueous phase may now be evaporated to recover a solidnitrogeneous product.

The invention is illustrated but not limited with reference to thefollowing examples:

EXAMPLE 1 25 kilograms of an aqueous yeast cream containing 15 percentby weight of the yeast Candida tropicaliswhich has been grown on gasoil, then washed with a nonionic detergent, was brought to pH 7.6 byaddition of a 2N soda solution; 270 grams of disodium phosphate (PO HNaH O) and 5.34 of citric acid (citric acid lH O) was added; thetemperature of the reaction mixture was brought to 70 C., when thistemperature was reached 15 grams of papain (Schwartz) and 15 grams ofcysteine hydrochloride was added; the temperature was kept at 70 C. foran hour;-the material was then centrifuged so that a residue and aliquid product were obtained; this residue was'washed with 8 liters ofdistilled water and recentrifugedThe resulting residue was taken up by15 liters of distilled water, the pH was adjusted to by adding a 2Nsolution of hydrochloric acid; 138 grams of disodium phosphate (PO HNal2l-i O) and 38.1 grams of citric acid (citric acid lH O) were added;this new mixture was heated and kept at 40 C. for 2 hours in thepresence of 25 grams of cellulase (B made by SEAB) and 25 grams oflipase B (Rohm and Haas). The product was centrifuged and the liquidproduct obtained wasadded to the liquid products previously obtained bycentrifuging. The mixture of liquids was dried by spray drying. 2.25kilograms of dry product containing 1 l percent by weight of nitrogenwas obtained.

EXAMPLE 2 10 kilograms of an aqueous suspension of bakers yeastcontaining 15 percent of dry material (Saccharomyces eerevisiae) werebrought to pH 7.6 by adding a 2N soda solution; then 3 liters of bufferat pH 7.6 (390 ml. of a 0.2N solution of monosodium phosphate and 2,6i0ml. of a 0.2M solution of disodium phosphate) were added, thetemperature of the suspension was brought to 70 C.; when thistemperature was reached 6 grams of Schwartz Ficine and 3 grams ofcysteine hydrochloride were added; this was left to digest for an hourand after centrifuging a liquid'product and a residue were obtained;this residue was washed with 3 liters of distilled water andrecentrifuged. The resulting residue was taken up by 3.3 liters of waterand brought to pH 5 by a 2N hydrochloric acid solution. This, suspensionwas mixed with 3.3 liters of phosphate buffer-citric acid, to give apH=5. The temperature was maintained at 40 C. in the presence of 15 g.of cellulase (SEAB) and 15 grams of lipase B (Rohm and Haas) for 2hours. The product was centrifuged and the liquid obtained was added tothe liquid products previously obtained by centrifuging. The mixture ofliquids was dried by spray drying. About 700 grams of dry productcontaining 10 percentby weight of nitrogen was obtained.

What we claim is:

l. A process which comprises treating a material with a proteolyticenzyme selected from the group consisting of pepsin, trypsin, bromelin,ficin and papain, such material essentially comprising cells of a yeast,said cells having a cell wall, constructed at least in part of at leastone compound having a carbohydrate component and a protein component,the treatment of said material being carried out whereby degeneration ofthe protein component takes place and whereby the carbohydratecomponents is rendered susceptible to enzymic attack, thereafterdeactivating or removing the proteolytic enzyme which is present in thecell walls, and further treating said material with an enzyme capable ofattacking a carbohydrate present in the cell wall, whereby degenerationof said carbohydrates takes place and thereafter recovering an aqueousfraction containing nitrogenous compounds.

2. A process according to claim 1 in which papain is employed in anamount constituting 02-03 percent of the dry cell weight of the yeast.

3. A process according to claim 1 in which the treated materialremaining after the treatment with the proteolytic enzyme is subjectedto phase separation.

4. A process according to claim 3 in which, after phase separation, theyeast-containing fraction is washed with an aqueous medium.

5. A process according to claim 1 in which the enzyme which is employedto attach carbohydrate of the cell is a cellulase.

6. A process according to claim 5 in which a lipase is employed with thecellulase.

7. A process according to claim A in which the yeast is CandidaLipalytica.

8. A process according to claim 1 in which the yeast is Candidatropicalis.

9. A process according to claim l in which the yeast has been cultivatedon a hydrocarbon-containing substrate.

Y 22%;?" UNITED SI'A'IES PA'HSNI' urnur; CERTIFICATE OF CORRECTIONPatentlio. 3,645,845 Dated February 29, 1972 Inventofla) Phi lippe Cortel I is certified that error appears in the above-identified patent andthat said Letters Patent are hereby corrected as shown below:

I Column 1, line 68, "IMRA" should read INRA Column 2, line 36, "sue"should read use Column 3, line 49, "(P0 HNa .H O)" should read (P0 HNa.l H20) and Claim 5, line 2, "attach" should read attack Signed andsealed this 11th day of July 1 972.

(SEAL) Attest:

- EDWARD M.FLETCHER, JR. ROBERT GOTTSCHALK Attesting OfficerCommissioner of Patents

2. A process according to claim 1 in which papain is employed in anamount constituting 0.2-0.3 percent of the dry cell weight of the yeast.3. A process according to claim 1 in which the treated materialremaining after the treatment with the proteolytic enzyme is subjectedto phase separation.
 4. A process according to claim 3 in which, afterphase separation, the yeast-containing fraction is washed with anaqueous medium.
 5. A process according to claim 1 in which the enzymewhich is employed to attach carbohydrate of the cell is a cellulase. 6.A process according to claim 5 in which a lipase is employed with thecellulase.
 7. A process according to claim 1 in which the yeast isCandida LipoLytica.
 8. A process according to claim 1 in which the yeastis Candida tropicalis.
 9. A process according to claim 1 in which theyeast has been cultivated on a hydrocarbon-containing substrate.